This study describes the effect of starch-synthesizing enzymes on biosynthesis of storage starch in rice amylose-extender mutants, which contain branched D-glucans with abnormal structures. Western blot analysis indicated that two out of five amylose-extender mutant lines lacked an isoform of starch branching enzyme, termed RBE3, although the levels of granule-bound starch synthase and a major form of branching enzyme, RBE1, were normal in these two mutants. Proteins corresponding to the 87-kDa RBE3 molecule were present in the three other amylose-extender mutants as well as in the wild type. However, the level of branching enzyme activity significantly decreased in all amylose-extender mutants, suggesting that the 87-kDa proteins in these three mutants are inactive forms of RBE3. Therefore, we conclude that formation of the abnormal branched glucans in the amylose-extender mutant of rice is due to the lack of the RBE3 activity. The cDNA clones encoding RBE3 have been identified from a normal rice seed cDNA library in lambda gt11, using a synthetic oligonucleotide as a probe. The deduced amino acid sequence of RBE3 indicates that this protein is initially synthesized as a precursor of 825 amino acids, including a 65-residue transit peptide at the NH2 terminus. The sequences of the catalytic regions in amylolytic enzymes are highly conserved in the sequence of RBE3. Thus, the branching enzyme isoform belongs to a family of the amylolytic enzymes. RBE3 also shares a noticeable degree of sequence identity with RBE1, especially at the central portion of the protein molecule. However, RBE3 possesses an approximately 70-residue extra sequence at the NH2 terminus and lacks a COOH-terminal sequence of almost 50 residues as compared with RBE1. The structural differences at both termini may explain the distinct role in starch synthesis for RBE1 and RBE3.